High performance liquid detection method of tubuterol

Abstract

The invention discloses a high performance liquid chromatography method for tubuterol, and the high performance liquid chromatography conditions are as follows: a chromatographic column is an octadecylsilane bonded phase chromatographic column, and the specification of the chromatographic column is 4.6 mm * 250mm, 5 [mu] m; a mobile phase: a mobile phase A is methanol, a mobile phase B is a phosphate buffer solution, the following gradient elution procedure is adopted, and the mobile phase ratio is 0-20 min, the mobile phase A is 25%-85%, and the mobile phase B is 75%-15%; 20-21 min, the mobile phase A accounts for 85%-25%, and the mobile phase B accounts for 15%-75%; the mobile phase A accounts for 25% and the mobile phase B accounts for 75% in 21-30 min; and the flow velocity of the mobile phase is 0.8-1.2 mL / min. According to the high performance liquid detection method, quantitative detection of the tobrabuterol content can be realized, and the detection method is high in specificity, sensitivity and accuracy. Meanwhile, according to the detection method, the determination time is saved, the operation is simple and convenient, the working efficiency is improved, and the detection cost is reduced.

Description

Technical Field

[0001] This application relates to the technical field of liquid chromatography analysis and detection, and particularly relates to a high performance liquid chromatography detection method for tulobuterol. Background Art

[0002] Tulobuterol, with the chemical name of 2-tert-butylamino-1-(2-chlorophenyl)ethanol, has the molecular formula C 12 H 18 ClNO, a molecular weight of 227.73, and a CAS number of 41570-61-0. Tulobuterol is a selective β2-adrenergic receptor agonist, mainly used for the treatment of respiratory diseases such as bronchial asthma and chronic obstructive pulmonary disease (COPD). The content determination methods for tulobuterol mainly include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), ultraviolet spectrophotometry (UV), etc.

[0003] High performance liquid chromatography (HPLC) is one of the most commonly used analytical methods in drug content detection. Its core advantages include: (1) high sensitivity and low detection limit. HPLC can detect drug concentrations in the range of ng / mL to μg / mL, suitable for raw materials, preparations, and low-dose biological samples (such as plasma and urine); (2) excellent selectivity. By using different chromatographic columns and mobile phases, different drug components can be effectively separated; (3) broad linear range. The standard curve of HPLC usually covers a linear detection range of 0.5 - 100 μg / mL, suitable for quantitative detection of samples with different concentrations. However, during the detection process of tulobuterol, it was found that when methanol and water were used as the mobile phase (v:v = 1:3), the chromatographic peaks were messy and had serious tailing. When Huang Lumin measured the content of tulobuterol by HPLC, using acetonitrile and 0.1% formic acid aqueous solution (v:v = 1:1), the peak shape of the chromatographic peak improved significantly but there was still tailing. Han Xiao et al. used acetonitrile and phosphate buffer solution as the mobile phase (v:v = 1:3), and the peak shape of the chromatographic peak was neat but there was also tailing. Peak tailing in liquid chromatography (such as HPLC) will directly affect the accuracy and reliability of the analysis results, leading to problems such as inaccurate quantification of tulobuterol detection, decreased resolution, decreased sensitivity, and poor reproducibility. At the same time, long-term tailing will reduce the service life of the chromatographic column.

[0004] Therefore, it is urgent to establish a high-efficiency, fast, and highly sensitive detection method for tulobuterol. Summary of the Invention

[0005] In view of the problems existing in the prior art, a high performance liquid chromatography detection method for tulobuterol of this application is proposed to overcome the above technical problems or at least partially solve the above technical problems.

[0006] To achieve the above object, the present application provides the following technical solutions:

[0007] An HPLC detection method for tulobuterol, and the HPLC conditions are as follows:

[0008] Chromatographic column: octadecylsilane-bonded phase chromatographic column (C18), the chromatographic column specification is 4.6mm * 250mm, 5μm;

[0009] Mobile phase: Mobile phase A is methanol, and mobile phase B is phosphate buffer solution. The following gradient elution program is adopted, and the proportions of the mobile phases are all volume percentages:

[0010] 0 - 20 min, mobile phase A is 25% - 85%, and mobile phase B is 75% - 15%;

[0011] 20 - 21 min, mobile phase A is 85% - 25%, and mobile phase B is 15% - 75%;

[0012] 21 - 30 min, mobile phase A is 25%, and mobile phase B is 75%;

[0013] Mobile phase flow rate: 0.8 - 1.2 mL / min;

[0014] Through the above detection method, the working efficiency of tulobuterol detection is significantly improved, and costs such as labor, time, and solvents are saved. Moreover, the detection sensitivity is high and the error is small.

[0015] Preferably, the concentration of the phosphate buffer solution is 0.1 mol / L.

[0016] More preferably, the pH of the phosphate buffer solution is 2.5 ± 0.1.

[0017] Most preferably, the phosphate buffer solution is a mixed solution of potassium monohydrogen phosphate and phosphoric acid.

[0018] Preferably, the mobile phase flow rate is 0.8 mL / min.

[0019] Preferably, the column temperature of the HPLC is 30 - 50 °C;

[0020] Preferably, the column temperature is 40 °C.

[0021] Preferably, the detection wavelength of the HPLC is 217 nm.

[0022] Preferably, the injection volume in the HPLC detection process is 10 μL.

[0023] Advantages of the present application:

[0024] Through reasonable method design, the method for detecting tulobuterol provided by this application has strong specificity, high sensitivity and accuracy compared with the prior art. At the same time, this method saves the determination time, is easy to operate, improves work efficiency and reduces the detection cost. Description of the Drawings

[0025] Figure 1 It is the standard working curve graph of tulobuterol provided by the embodiment of this application.

[0026] Figure 2 It is the high performance liquid chromatography (HPLC) spectrum of sample 1 in Embodiment 1 of this application.

[0027] Figure 3 It is the high performance liquid chromatography (HPLC) spectrum of sample 2 in Embodiment 2 of this application.

[0028] Figure 4 It is the high performance liquid chromatography (HPLC) spectrum of sample 3 in Embodiment 3 of this application.

[0029] Figure 5 It is the high performance liquid chromatography (HPLC) spectrum of sample 4 in Embodiment 4 of this application. Detailed Embodiments

[0030] In order to make the objectives, technical solutions and advantages of this application clearer, the following further details this application in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0031] Those skilled in the art of this technology can understand that unless specifically stated, the "the", "this", "the above", etc. used in the text of this application can also include the plural form.

[0032] Those skilled in the art of this technology can understand that for those experimental steps or conditions not specified in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed; for those raw materials or instrument equipment without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] Those skilled in the art of this technology can understand that unless otherwise stated in this application, when the embodiments give a numerical range, any value at both ends of each numerical range and any value between the two ends can be selected. Unless otherwise defined, all the technical and scientific terms used in this application, based on the understanding of those skilled in the art of this technology of the prior art and the description of this application, can also use any methods, equipment and materials of the prior art similar or equivalent to those described in the embodiments of this application to implement this application.

[0034] The main reagents and instruments used in the embodiments of this application are as follows:

[0035] Standard: Tulobuterol standard (mass fraction > 99%)

[0036] Reagents: Methanol (chromatographic grade, Bidepharm), phosphate solution (analytical grade, Bidepharm), phosphoric acid (chromatographic grade, Aladdinpharm), and the experimental water meets the specification of grade 3 water in GB / T 6682 - 2008 (distilled water or deionized water or water of equivalent purity)

[0037] Instruments: Shimadzu LC - 20AT high - performance liquid chromatograph equipped with a diode array detector; Analytical balance TD102 (Tianjin Tianma Instrument Factory); PHS - 25 Leici digital pH meter (Shanghai Precision Scientific Instrument Co., Ltd.); Ultrasonic cleaner; Vortex oscillator

[0038] Example 1

[0039] This example provides a high - performance liquid chromatography detection method for tulobuterol, and the specific steps are as follows:

[0040] (1) Preparation of phosphate buffer solution

[0041] Weigh 1.36 g of potassium hydrogen phosphate, add water to 2000 mL, and then adjust the pH to 2.5 with phosphoric acid. The obtained phosphate buffer solution is used as mobile phase B

[0042] (2) Preparation of tulobuterol standard stock solution

[0043] Weigh 400 mg of tulobuterol, dissolve it with methanol and make up the volume to 100 mL to obtain a 4.0 g / L tulobuterol standard stock solution; respectively pipette 2.5 mL, 5 mL, 7.5 mL, 10 mL of the tulobuterol standard stock solution into 10 - mL volumetric flasks, dilute with water to the scale, shake well and reserve. Tulobuterol standard solutions with mass concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL are obtained

[0044] (3) Preparation of tulobuterol standard working curve

[0045] Arrange the tulobuterol standard solutions in ascending order of mass concentration and measure their response values under the specified high - performance liquid chromatography instrument conditions. Among them, the high - performance liquid chromatography conditions are as follows:

[0046] Chromatographic column: Octadecylsilane - bonded silica gel chromatographic column (C18), the chromatographic column specification is 4.6 mm * 250 mm, 5 μm

[0047] Mobile phase: Mobile phase A is methanol, and mobile phase B is 0.1 mol / L phosphate buffer solution. The following gradient elution program is adopted, where the mobile phase ratios are all volume percentages:

[0048] 0 - 20 min, mobile phase A is 25% - 85%, mobile phase B is 75% - 15%;

[0049] 20 - 21 min, mobile phase A is 85% - 25%, mobile phase B is 15% - 75%;

[0050] 21 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0051] Mobile phase flow rate: 0.8 mL / min;

[0052] Column temperature: 40 °C;

[0053] Injection volume: 10 μL

[0054] Detection wavelength: 217 nm.

[0055] Using the working solution mass concentration as the abscissa and the corresponding total peak area as the ordinate to make a standard curve, as Figure 1 shown, the standard curve equation is y = 11496749.70x + 12636892.50, R 2 = 0.99.

[0056] (4) Sample treatment:

[0057] Take 1 mL of 1 mg / mL tulobuterol standard solution and place it in a 10 mL volumetric flask. Dilute and make up the volume with water, then vortex and mix well with a vortex oscillator, and filter through a 0.45 μm filter membrane to obtain the first sample to be tested. Detect the first sample by high performance liquid chromatography, and its high performance liquid chromatography conditions are the same as those in step (3).

[0058] Figure 2 is the high performance liquid chromatography chart of the first sample, and the tulobuterol content of the first sample is measured to be 99.81%.

[0059] In addition, calculate the tulobuterol content in the first sample through the standard curve, and the calculation formula is as follows:

[0060]

[0061] X'represents the total tulobuterol content, with the unit of %; ρ represents the total mass concentration of tulobuterol in the sample calculated through the standard curve, with the unit of mg / L; V represents the sample volume made up, with the unit of mL; m represents the sample sampling amount, with the unit of g. According to Figure 2 the peak area shown and substituting it into the standard curve and combining the above formula, the tulobuterol content in the first sample is calculated to be 100.7%.

[0062] From the above test results, it can be seen that the difference between the test result of Sample 1 by the high performance liquid chromatography method of this application and the value obtained through the standard curve is only 0.89%, indicating that the high performance liquid chromatography method for tulobuterol provided in this example has high accuracy.

[0063] Example 2

[0064] This example provides a high performance liquid chromatography method for tulobuterol, and its specific steps are as follows:

[0065] Take 1 mL of 1 mg / mL tulobuterol standard solution and place it in a 10 mL volumetric flask. Dilute it to the mark with water, then mix it evenly with a vortex oscillator and filter it through a 0.45 μm filter membrane to obtain Test Sample 2. Detect Test Sample 2 by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0066] Chromatographic column: Octadecylsilane bonded phase chromatographic column (C18), the chromatographic column specification is 4.6 mm * 250 mm, 5 μm;

[0067] Mobile phase: Mobile phase A is methanol, and mobile phase B is 0.1 mol / L phosphate buffer solution (the same as in Example 1). The following gradient elution program is adopted, and the mobile phase ratios are all volume percentages:

[0068] 0 - 20 min, mobile phase A is 25% - 85%, and mobile phase B is 75% - 15%;

[0069] 20 - 21 min, mobile phase A is 85% - 25%, and mobile phase B is 15% - 75%;

[0070] 21 - 30 min, mobile phase A is 25%, and mobile phase B is 75%;

[0071] Mobile phase flow rate: 1.0 mL / min;

[0072] Column temperature: 40 °C;

[0073] Injection volume: 10 μL

[0074] Detection wavelength: 217 nm.

[0075] Figure 3 is the high performance liquid chromatography chart of Test Sample 2, and the measured tulobuterol content of Test Sample 2 is 99.83%.

[0076] Example 3

[0077] This example provides a high performance liquid chromatography method for tulobuterol, and its specific steps are as follows:

[0078] Take 1 mL of the tobuterol standard solution at 1 mg / mL, place it in a 10-mL volumetric flask, dilute it to the mark with water, then vortex and mix well, filter through a 0.45-μm filter membrane to obtain the sample to be tested, sample three. Detect sample three by high-performance liquid chromatography. The high-performance liquid chromatography conditions are as follows:

[0079] Chromatographic column: octadecylsilane-bonded phase chromatographic column (C18), the chromatographic column specifications are 4.6 mm * 250 mm, 5 μm;

[0080] Mobile phase: mobile phase A is methanol, mobile phase B is 0.1 mol / L phosphate buffer solution (same as in Example 1). The following gradient elution program is adopted, where the mobile phase ratios are all volume percentages:

[0081] 0 - 20 min, mobile phase A is 25% - 85%, mobile phase B is 75% - 15%;

[0082] 20 - 21 min, mobile phase A is 85% - 25%, mobile phase B is 15% - 75%;

[0083] 21 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0084] Mobile phase flow rate: 1.2 mL / min;

[0085] Column temperature: 30 °C;

[0086] Injection volume: 10 μL

[0087] Detection wavelength: 217 nm.

[0088] Figure 4 The high-performance liquid chromatography diagram of sample three shows that the tobuterol content in sample three is 99.89%.

[0089] Example 4

[0090] This example provides a high-performance liquid chromatography detection method for tobuterol. The specific steps are as follows:

[0091] Take 1 mL of the tobuterol standard solution at 1 mg / mL, place it in a 10-mL volumetric flask, dilute it to the mark with water, then vortex and mix well, filter through a 0.45-μm filter membrane to obtain the sample to be tested, sample three. Detect sample three by high-performance liquid chromatography. The high-performance liquid chromatography conditions are as follows:

[0092] Chromatographic column: octadecylsilane-bonded phase chromatographic column (C18), the chromatographic column specifications are 4.6 mm * 250 mm, 5 μm;

[0093] Mobile phase: Mobile phase A is methanol, and mobile phase B is 0.1 mol / L phosphate buffer solution (the same as in Example 1). The following gradient elution program is adopted, where the proportions of the mobile phases are all in volume percentages:

[0094] 0 - 20 min, mobile phase A is 25% - 85%, and mobile phase B is 75% - 15%;

[0095] 20 - 21 min, mobile phase A is 85% - 25%, and mobile phase B is 15% - 75%;

[0096] 21 - 30 min, mobile phase A is 25%, and mobile phase B is 75%;

[0097] Flow rate of the mobile phase: 0.8 mL / min;

[0098] Column temperature: 50 °C;

[0099] Sample injection volume: 10 μL

[0100] Detection wavelength: 217 nm.

[0101] Figure 5 This is the high - performance liquid chromatography (HPLC) chromatogram of Sample 4. The measured content of tulobuterol in Sample 4 is 99.9%.

[0102] Comparative Example 1

[0103] This comparative example provides a high - performance liquid chromatography (HPLC) detection method for tulobuterol. The specific steps are as follows:

[0104] Take 1 mL of 1 mg / mL tulobuterol standard solution and place it in a 10 - mL volumetric flask. Dilute it to the mark with water, then mix it well with a vortex oscillator and filter it through a 0.45 - μm filter membrane to obtain the sample to be tested, Sample 5. The sample 5 is detected by high - performance liquid chromatography. The high - performance liquid chromatography conditions are as follows:

[0105] Chromatographic column: Octadecylsilane - bonded silica gel column (C18), the column specification is 4.6 mm * 250 mm, 5 μm;

[0106] Mobile phase: Mobile phase A is methanol, and mobile phase B is water, where the proportions of the mobile phases are all in volume percentages:

[0107] 0 - 30 min, mobile phase A is 25%, and mobile phase B is 75%;

[0108] Flow rate of the mobile phase: 0.8 mL / min;

[0109] Column temperature: 40 °C;

[0110] Sample injection volume: 10 μL

[0111] Detection wavelength: 217 nm.

[0112] Comparative Example 2

[0113] This comparative example provides a high performance liquid chromatography (HPLC) detection method for tulobuterol, and the specific steps are as follows:

[0114] Take 1 mL of 1 mg / mL tulobuterol standard solution, place it in a 10 mL volumetric flask, dilute it to the mark with water, then mix it evenly with a vortex oscillator, filter it through a 0.45 μm filter membrane to obtain sample VI for testing. The sample VI is detected by high performance liquid chromatography, and the HPLC conditions are as follows:

[0115] Chromatographic column: Octadecylsilane bonded silica gel column (C18), the chromatographic column specification is 4.6 mm * 250 mm, 5 μm;

[0116] Mobile phase: Mobile phase A is methanol, and mobile phase B is water. The proportion of the mobile phase is in volume percentage:

[0117] From 0 to 30 min, mobile phase A is 25%, and mobile phase B is 75%;

[0118] Mobile phase flow rate: 1.0 mL / min;

[0119] Column temperature: 40 °C;

[0120] Injection volume: 10 μL

[0121] Detection wavelength: 217 nm.

[0122] Comparative Example 3

[0123] This comparative example provides a high performance liquid chromatography (HPLC) detection method for tulobuterol, and the specific steps are as follows:

[0124] Take 1 mL of 1 mg / mL tulobuterol standard solution, place it in a 10 mL volumetric flask, dilute it to the mark with water, then mix it evenly with a vortex oscillator, filter it through a 0.45 μm filter membrane to obtain sample VII for testing. The sample VII is detected by high performance liquid chromatography, and the HPLC conditions are as follows:

[0125] Chromatographic column: Octadecylsilane bonded silica gel column (C18), the chromatographic column specification is 4.6 mm * 250 mm, 5 μm;

[0126] Mobile phase: Mobile phase A is methanol, and mobile phase B is water. The proportion of the mobile phase is in volume percentage:

[0127] From 0 to 30 min, mobile phase A is 25%, and mobile phase B is 75%;

[0128] Mobile phase flow rate: 1.2 mL / min;

[0129] Column temperature: 40 °C;

[0130] Sample injection volume: 10 μL

[0131] Detection wavelength: 217 nm.

[0132] Comparative Example 4

[0133] This comparative example provides a high performance liquid chromatography (HPLC) detection method for tulobuterol. The specific steps are as follows:

[0134] Take 1 mL of 1 mg / mL tulobuterol standard solution and place it in a 10 mL volumetric flask. Dilute it to the mark with water, then vortex and mix well with a vortex oscillator. Filter through a 0.45 μm filter membrane to obtain the sample to be tested, Sample VIII. Detect Sample VIII by high performance liquid chromatography. The HPLC conditions are as follows:

[0135] Chromatographic column: Octadecylsilane bonded phase chromatographic column (C18), column specification: 4.6 mm * 250 mm, 5 μm;

[0136] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is 0.1 mol / L phosphate buffer solution (the same as in Example 1). The proportions of the mobile phases are all in volume percentages:

[0137] 0 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0138] Mobile phase flow rate: 0.8 mL / min;

[0139] Column temperature: 40 °C;

[0140] Sample injection volume: 10 μL

[0141] Detection wavelength: 217 nm.

[0142] Comparative Example 5

[0143] This comparative example provides a high performance liquid chromatography (HPLC) detection method for tulobuterol. The specific steps are as follows:

[0144] Take 1 mL of 1 mg / mL tulobuterol standard solution and place it in a 10 mL volumetric flask. Dilute it to the mark with water, then vortex and mix well with a vortex oscillator. Filter through a 0.45 μm filter membrane to obtain the sample to be tested, Sample IX. Detect Sample IX by high performance liquid chromatography. The HPLC conditions are as follows:

[0145] Chromatographic column: Octadecylsilane bonded phase chromatographic column (C18), column specification: 4.6 mm * 250 mm, 5 μm;

[0146] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is 0.1 mol / L phosphate buffer solution (the same as in Example 1). The proportions of the mobile phases are all in volume percentages:

[0147] 0 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0148] Flow rate of mobile phase: 1.0 mL / min;

[0149] Column temperature: 40 °C;

[0150] Injection volume: 10 μL

[0151] Detection wavelength: 217 nm.

[0152] Comparative Example 6

[0153] This comparative example provides a high - performance liquid chromatography detection method for tulobuterol, and its specific steps are as follows:

[0154] Take 1 mL of 1 mg / mL tulobuterol standard solution, place it in a 10 mL volumetric flask, dilute it to the mark with water, then vortex and mix well with a vortex oscillator, filter through a 0.45 μm filter membrane to obtain sample X, and detect sample X by high - performance liquid chromatography. The high - performance liquid chromatography conditions are as follows:

[0155] Chromatographic column: octadecylsilane - bonded phase chromatographic column (C18), the chromatographic column specification is 4.6 mm * 250 mm, 5 μm;

[0156] Mobile phase: mobile phase A is acetonitrile, mobile phase B is 0.1 mol / L phosphate buffer solution (the same as in Example 1), and the proportion of the mobile phase is in volume percentage:

[0157] 0 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0158] Flow rate of mobile phase: 1.2 mL / min;

[0159] Column temperature: 40 °C;

[0160] Injection volume: 10 μL

[0161] Detection wavelength: 217 nm.

[0162] Comparative Example 7

[0163] This comparative example provides a high - performance liquid chromatography detection method for tulobuterol, and its specific steps are as follows:

[0164] Take 1 mL of 1 mg / mL tulobuterol standard solution, place it in a 10 mL volumetric flask, dilute it to the mark with water, then vortex and mix well with a vortex oscillator, filter through a 0.45 μm filter membrane to obtain sample XI, and detect sample XI by high - performance liquid chromatography. The high - performance liquid chromatography conditions are as follows:

[0165] Chromatographic column: Octadecylsilyl-bonded silica gel column (C18), column specification: 4.6mm * 250mm, 5μm;

[0166] Mobile phase: Mobile phase A is methanol, mobile phase B is 0.1mol / L phosphate buffer solution (same as Example 1), and the following gradient elution program is adopted, where the mobile phase ratios are all volume percentages:

[0167] 0 - 20 min, mobile phase A is 25% - 85%, mobile phase B is 75% - 15%;

[0168] 20 - 21 min, mobile phase A is 85% - 25%, mobile phase B is 15% - 75%;

[0169] 21 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0170] Mobile phase flow rate: 0.7 mL / min;

[0171] Column temperature: 40°C;

[0172] Injection volume: 10 μL

[0173] Detection wavelength: 217 nm.

[0174] Comparative Example 8

[0175] This comparative example provides a high performance liquid chromatography detection method for tulobuterol, and its specific steps are as follows:

[0176] Take 1 mL of 1 mg / mL tulobuterol standard solution, place it in a 10 mL volumetric flask, dilute and make up the volume with water, then vortex and mix well with a vortex oscillator, filter through a 0.45 μm filter membrane to obtain the sample twelve to be detected, and detect the sample twelve by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0177] Chromatographic column: Octadecylsilyl-bonded silica gel column (C18), column specification: 4.6mm * 250mm, 5μm;

[0178] Mobile phase: Mobile phase A is methanol, mobile phase B is 0.1mol / L phosphate buffer solution (same as Example 1), and the following gradient elution program is adopted, where the mobile phase ratios are all volume percentages:

[0179] 0 - 20 min, mobile phase A is 25% - 85%, mobile phase B is 75% - 15%;

[0180] 20 - 21 min, mobile phase A is 85% - 25%, mobile phase B is 15% - 75%;

[0181] 21 - 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0182] Mobile phase flow rate: 1.3 mL / min;

[0183] Column temperature: 30 °C;

[0184] Sample injection volume: 10 μL

[0185] Detection wavelength: 217 nm.

[0186] The high performance liquid chromatography detection results provided by the above examples and comparative examples are shown in Table 1 below.

[0187] Table 1

[0188]

[0189] As can be seen from the results in Table 1, the high performance liquid chromatography detection methods of Examples 1-4 can accurately and highly sensitively detect the content of tulobuterol in the sample. In Comparative Examples 1-3, the mobile phase of methanol:water = 1:3 (v:v) was used, and the peak shapes of the liquid chromatography were messy and severely tailed. In Comparative Examples 4-6, the mobile phase of acetonitrile:phosphate buffer solution = 1:3 (v:v) was used, and although the peak shapes of the liquid chromatography were neat, different degrees of tailing occurred. In Comparative Examples 7 and 8, although the same mobile phase as in Examples 1-4 was used, due to too low or too high flow rates, different degrees of tailing also occurred in the peak shapes of the liquid chromatography.

[0190] In summary, the high performance liquid chromatography method for detecting the content of tulobuterol provided by the embodiments of the present application has screened out a highly specific tulobuterol detection method by optimizing parameters such as the mobile phase, elution program, and flow rate. This method has high detection sensitivity and accuracy. At the same time, this method saves the determination time, is easy to operate, improves work efficiency and reduces the detection cost.

[0191] The above has introduced the present application in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present application. The description of the above examples is only used to help understand the present application and its core idea. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present application, several improvements and modifications can still be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. An HPLC detection method for tulobuterol, characterized in that, The high performance liquid chromatography conditions are as follows: Chromatographic column: octadecylsilyl bonded phase chromatographic column, with a chromatographic column specification of 4.6 mm * 250 mm, 5 μm; Mobile phase: Mobile phase A is methanol, and mobile phase B is phosphate buffer solution. The following gradient elution program is adopted, where the mobile phase ratios are all volume percentages: 0 - 20 min, mobile phase A is 25% - 85%, and mobile phase B is 75% - 15%; 20 - 21 min, mobile phase A is 85% - 25%, and mobile phase B is 15% - 75%; 21 - 30 min, mobile phase A is 25%, and mobile phase B is 75%; Mobile phase flow rate: 0.8 - 1.2 mL / min.

2. The high performance liquid chromatography detection method according to claim 1, characterized in that The concentration of the phosphate buffer solution is 0.1 mol / L.

3. The high performance liquid chromatography detection method according to claim 2, wherein The pH of the phosphate buffer solution is 2.5 ± 0.

1.

4. The high performance liquid chromatography detection method according to claim 3, wherein The phosphate buffer solution is a mixed solution of potassium hydrogen phosphate and phosphoric acid.

5. The high performance liquid chromatography detection method according to claim 1, wherein The mobile phase flow rate is 0.8 mL / min.

6. The high-performance liquid detection method according to claim 1, characterized in that The column temperature of the high performance liquid chromatography is 30 - 50 °C.

7. The high performance liquid chromatography detection method according to claim 6, characterized in that The column temperature is 40 °C.

8. The high performance liquid chromatography detection method according to claim 1, characterized in that The detection wavelength of the high performance liquid chromatography is 217 nm.

9. The high performance liquid chromatography detection method according to claim 1, characterized in that The injection volume during the high performance liquid detection process is 10 μL.

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